The present invention relates to automated devices for maintaining an even dispersion of solid phase extraction beads in small volume tubes.
Beads are used in an ever increasing number of applications including separation, detection, and nanotechnology research. For biological assays or for target concentration, a capture molecule such as, but not limited to, an antibody or oligonucleotide is immobilized on the beads, and the modified beads are used to capture the target from a sample fluid. Effective dispersion of the beads is important both during the immobilization procedure and during the target capture reaction. The beads are usually a polymer, metal colloid, glass, or magnetic material.
Solid-phase extraction has become popular as a laboratory tool for bio-separation and target pre-concentration. Beads packed into columns are used for initial sample cleanup prior to analysis using gas chromatography and GC-MS, HPLC, and LC-MS systems. In addition to bench-top applications, solid phase extraction columns have been incorporated into microfluidics-format portable devices as well as larger, automated, high throughput systems for bio-separations in clinical chemistry, pharmaceutical bio-analysis, forensics, drug discovery, and analytical biochemistry.
Recently, single tube techniques based on free-standing suspensions of beads have been introduced; these are especially useful for quickly and efficiently capturing specific or random nucleic acid fragments in low volume tubes. Bead-based technologies of this type replace more labor intensive precipitation reactions and eliminate the use of phenols and other toxic chemicals.
Although the introduction of free-standing bead suspensions has produced a significant decrease in the time and effort required for solid phase extractions, the suggested procedures are still far from automated. It is desirable to eliminate as many manual steps as possible in order to minimize the time and labor, to enhance reproducibility, and to integrate the processes into automated sensors and reactors.
Several attributes are desirable in this type of automated device. First, it can be able to maintain an even dispersion of beads in solution, in order to maximize exposure of the bead surface area to components in solution. Second, it can continuously mix the beads for extended time periods in order to avoid gradients in solution, while avoiding the formation of droplets or bubbles in solution. Third, the device can have the capability of performing this agitation at a wide variety of temperatures. Fourth, the device can operate in a manner that provides reproducible results. Fifth, the device can be able to handle multiple samples simultaneously. Finally, the device can incorporate a mechanism for pelleting the beads (if possible), so that the supernatant can be removed from the reaction chamber.
During both attachment of the capture molecules and the use of the beads to bind the target(s), even dispersion of the beads is desirable so that there is maximum exposure of the bead surface to the solution. The formation of bubbles within the liquid or droplets on the inside walls of the tube is undesirable because it either causes decreased immobilization of the capture molecule or results in decreased exposure of the target to the capture molecule on the beads.
The current method of achieving even bead dispersion is to manually hold the tube in one hand and tap gently on the outside of the tube with the tip of the fingers of the other hand, or to place the tube in a vortex mixer. This method is limiting in several ways: First, the method requires a person to be present to actively tap the tube or hold it in a vortex mixer. This means that, for dispersion to be maintained, that person must continuously tap the tube. Usually, instead of doing this for extended periods of time, the person will return during the course of an experiment to disperse the beads once every few minutes. The result is that the beads do not remain uniformly dispersed throughout the experiment, but rather settle and are re-dispersed multiple times. Second, the method only allows one PCR tube at a time to undergo bead dispersion, even though multiple tubes may be needed for an experiment. Third, the method may cause the formation of unwanted bubbles or droplets. Fourth, the method is inconsistent between implementations. This means that results of an experiment that relies on even bead dispersion may be affected by the different degrees of dispersion delivered to different samples.
Another method of keeping beads dispersed is to agitate the tube containing the beads on some sort of electrical shaking device. This method eliminates the need for a person to continuously be present, but the method is limited in that it causes the formation of droplets and air bubbles which may interfere with binding. When agitation is finished, the tube containing the beads may need to be centrifuged to cause any droplets to settle. This requires a power source for the centrifuge, and it requires a separate piece of equipment (the centrifuge). Also, for single tubes, counterbalancing of the centrifuge might be required.
Using magnetic beads provides for simple isolation of the beads using a magnet whereas the nonmagnetic beads are usually captured by gravity sedimentation, centrifugation, or filtration. Once the beads are concentrated into a tightly packed pellet, the remaining supernatant may be removed or saved, depending on the objective of the operation. The nonmagnetic beads may take longer to separate from the sample fluids and the operations may be technically time consuming or inefficient, especially for high viscosity samples or very small beads.
A current method for settling magnetic beads is to hold the tube containing the beads upright over a magnet. This method is limited because it requires the presence of a separate piece of equipment (a strong magnet) and because the operator may be required to hold the tube for extended periods until the beads settle (e.g. because conical tubes will not sit upright and the bottom of the tube must be in close proximity to the magnet for the desired effect).
Another method of separating magnetic beads involves the use of a magnetic stand specifically designed for the purpose which usually places the magnet alongside the tube to produce a shorter path length. The beads are held on the side of the tube while the operator withdraws the fluid. This method is limited in that it requires a separate piece of equipment (the magnetic stand).